Genome-wide association studies reveal novel loci for resistance to groundnut rosette disease in the African core groundnut collection.

KEY MESSAGE: We identified markers associated with GRD resistance after screening an Africa-wide core collection across three seasons in Uganda Groundnut is cultivated in several African countries where it is a major source of food, feed and income. One of the major constraints to groundnut production in Africa is groundnut rosette disease (GRD), which is caused by a complex of three agents: groundnut rosette assistor luteovirus, groundnut rosette umbravirus and its satellite RNA. Despite several years of breeding for GRD resistance, the genetics of the disease is not fully understood. The objective of the current study was to use the African core collection to establish the level of genetic variation in their response to GRD, and to map genomic regions responsible for the observed resistance. The African groundnut core genotypes were screened across two GRD hotspot locations in Uganda (Nakabango and Serere) for 3 seasons. The Area Under Disease Progress Curve combined with 7523 high quality SNPs were analyzed to establish marker-trait associations (MTAs). Genome-Wide Association Studies based on Enriched Compressed Mixed Linear Model detected 32 MTAs at Nakabango: 21 on chromosome A04, 10 on B04 and 1 on B08. Two of the significant markers were localised on the exons of a putative TIR-NBS-LRR disease resistance gene on chromosome A04. Our results suggest the likely involvement of major genes in the resistance to GRD but will need to be further validated with more comprehensive phenotypic and genotypic datasets. The markers identified in the current study will be developed into routine assays and validated for future genomics-assisted selection for GRD resistance in groundnut.

Genetic fingerprinting and aflatoxin production of Aspergillus section Flavi associated with groundnut in eastern Ethiopia.

BACKGROUND: Aspergillus species cause aflatoxin contamination in groundnut kernels, being a health threat in agricultural products and leading to commodity rejection by domestic and international markets. Presence of Aspergillus flavus and A. parasiticus colonizing groundnut in eastern Ethiopia, as well as presence of aflatoxins have been reported, though in this region, no genetic studies have been done of these species in relation to their aflatoxin production. RESULTS: In this study, 145 Aspergillus isolates obtained from groundnut kernels in eastern Ethiopia were genetically fingerprinted using 23 Insertion/Deletion (InDel) markers within the aflatoxin-biosynthesis gene cluster (ABC), identifying 133 ABC genotypes. Eighty-four isolates were analyzed by Ultra-Performance Liquid Chromatography (UPLC) for in vitro aflatoxin production. Analysis of genetic distances based on the approximately 85 kb-ABC by Neighbor Joining (NJ), 3D-Principal Coordinate Analysis (3D-PCoA), and Structure software, clustered the isolates into three main groups as a gradient in their aflatoxin production. Group I, contained 98% A. flavus, including L- and non-producers of sclerotia (NPS), producers of B1 and B2 aflatoxins, and most of them collected from the lowland-dry Babile area. Group II was a genetic admixture population of A. flavus (NPS) and A. flavus S morphotype, both low producers of aflatoxins. Group III was primarily represented by A. parasiticus and A. flavus S morphotype isolates both producers of B1, B2 and G1, G2 aflatoxins, and originated from the regions of Darolabu and Gursum. The highest in vitro producer of aflatoxin B1 was A. flavus NPS N1436 (77.98 µg/mL), and the highest producer of aflatoxin G1 was A. parasiticus N1348 (50.33 µg/mL), these isolates were from Gursum and Darolabu, respectively. CONCLUSIONS: To the best of our knowledge, this is the first study that combined the use of InDel fingerprinting of the ABC and corresponding aflatoxin production capability to describe the genetic diversity of Aspergillus isolates from groundnut in eastern Ethiopia. Three InDel markers, AFLC04, AFLC08 and AFLC19, accounted for the main assignment of individuals to the three Groups; their loci corresponded to aflC (pksA), hypC, and aflW (moxY) genes, respectively. Despite InDels within the ABC being often associated to loss of aflatoxin production, the vast InDel polymorphism observed in the Aspergillus isolates did not completely impaired their aflatoxin production in vitro.

Genetic relationships among seven sections of genus Arachis studied by using SSR markers.

BACKGROUND: The genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x = 20) and the tetraploid species (2n = 2x = 40) are found in sections Arachis, Extranervosae and Rhizomatosae. Diploid species have great potential to be used as resistance sources for agronomic traits like pests and diseases, drought related traits and different life cycle spans. Understanding of genetic relationships among wild species and between wild and cultivated species will be useful for enhanced utilization of wild species in improving cultivated germplasm. The present study was undertaken to evaluate genetic relationships among species (96 accessions) belonging to seven sections of Arachis by using simple sequence repeat (SSR) markers developed from Arachis hypogaea genomic library and gene sequences from related genera of Arachis. RESULTS: The average transferability rate of 101 SSR markers tested to section Arachis and six other sections was 81% and 59% respectively. Five markers (IPAHM 164, IPAHM 165, IPAHM 407a, IPAHM 409, and IPAHM 659) showed 100% transferability. Cluster analysis of allelic data from a subset of 32 SSR markers on 85 wild and 11 cultivated accessions grouped accessions according to their genome composition, sections and species to which they belong. A total of 109 species specific alleles were detected in different wild species, Arachis pusilla exhibited largest number of species specific alleles (15). Based on genetic distance analysis, the A-genome accession ICG 8200 (A. duranensis) and the B-genome accession ICG 8206 (A. ipaënsis) were found most closely related to A. hypogaea. CONCLUSION: A set of cross species and cross section transferable SSR markers has been identified that will be useful for genetic studies of wild species of Arachis, including comparative genome mapping, germplasm analysis, population genetic structure and phylogenetic inferences among species. The present study provides strong support based on both genomic and genic markers, probably for the first time, on relationships of A. monticola and A. hypogaea as well as on the most probable donor of A and B-genomes of cultivated groundnut.

A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.).

BACKGROUND: Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers. RESULTS: A total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (< or =1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with > or = 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries. CONCLUSION: Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species.

AFLP-based molecular characterization of an elite germplasm collection of Jatropha curcas L., a biofuel plant.

Amplified fragment length polymorphism (AFLP) was employed to assess the diversity in the elite germplasm collection of Jatropha curcas, which has gained tremendous significance as a biofuel plant in India and many other countries recently. Forty-eight accessions, collected from six different states of India, were used with seven AFLP primer combinations that generated a total of 770 fragments with an average of 110 fragments per primer combination. A total of 680 (88%) fragments showed polymorphism in the germplasm analyzed, of which 59 (8.7%) fragments were unique (accession specific) and 108 (15.9%) fragments were rare (present in less than 10% accessions). In order to assess the discriminatory power of seven primer combinations used, a variety of marker attributes like polymorphism information content (PIC), marker index (MI) and resolving power (RP) values were calculated. Although the PIC values ranged from 0.20 (E-ACA/M-CAA) to 0.34 (E-ACT/M-CTT) with an average of 0.26 per primer combination and the MI values were observed in the range of 17.60 (E-ACA/M-CAA) to 32.30 (E-ACT/M-CTT) with an average of 25.13 per primer combination, the RP was recognized the real attribute for AFLP to determine the discriminatory power of the primer combination. The RP values for different primer combinations varied from 23.11 (E-ACA/M-CAA) to 46.82 (E-ACT/M-CTT) with an average of 35.21. Genotyping data obtained for all 680 polymorphic fragments were used to group the accessions analyzed using the UPGMA-phenogram and principal component analysis (PCA). Majority of groups obtained in phenogram and PCA contained accessions as per geographical locations. In general, accessions coming from Andhra Pradesh were found diverse as these were scattered in different groups, whereas accessions coming from Chhattisgarh showed occurrence of higher number of unique/rare fragments. Molecular diversity estimated in the present study combined with the datasets on other morphological/agronomic traits will be very useful for selecting the appropriate accessions for plant improvement through conventional as well as molecular breeding approaches.

Peanut Consumption in Malawi: An Opportunity for Innovation.

Peanuts are a valuable source of nutrients, but peanut consumption patterns, consumption frequencies, and the factors influencing peanut consumption in Malawi are not known. This study surveyed consumers to fill this knowledge gap and to assess Malawian consumers' readiness to try new food products. Out of the 489 respondents surveyed, all but three consumed peanuts (in any form). The majority (70.4%) consumed peanuts at least three times in a week. Chi-square test showed that demographic and socioeconomic variables had significant effects (p < 0.05) on peanut product preferences, the frequency of peanut consumption, and readiness to try new foods. For instance, women mostly preferred peanut flour compared to men, and peanut butter was the most preferred form for younger consumers. Logistic regression analysis showed that consumers with high school education or below were 2.35 times more likely to eat peanuts more often than consumers with post high school education. Among the participants that were ready to try new foods (54%), men and those with post high school education were 1.90 and 2.74 times more likely to try new foods than their respective counterparts. In general, the diversity of peanut products on the Malawian market is limited, and socioeconomic restrictions override consumer preferences. Therefore, future peanut-based food products innovations should explore ways to overcome such restrictions.

Advances in cereal genomics and applications in crop breeding.

Recent advances in cereal genomics have made it possible to analyse the architecture of cereal genomes and their expressed components, leading to an increase in our knowledge of the genes that are linked to key agronomically important traits. These studies have used molecular genetic mapping of quantitative trait loci (QTL) of several complex traits that are important in breeding. The identification and molecular cloning of genes underlying QTLs offers the possibility to examine the naturally occurring allelic variation for respective complex traits. Novel alleles, identified by functional genomics or haplotype analysis, can enrich the genetic basis of cultivated crops to improve productivity. Advances made in cereal genomics research in recent years thus offer the opportunities to enhance the prediction of phenotypes from genotypes for cereal breeding.

Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping.

BACKGROUND: With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. RESULTS: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. CONCLUSION: A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/gt-bt/lims/lims.asp.

Aspergillus and aflatoxin in groundnut (Arachis hypogaea L.) and groundnut cake in Eastern Ethiopia.

This study was conducted to assess major Aspergillus species and aflatoxins associated with groundnut seeds and cake in Eastern Ethiopia and evaluate growers' management practices. A total of 160 groundnut seed samples from farmers' stores and 50 groundnut cake samples from cafe and restaurants were collected. Fungal isolation was done from groundnut seed samples. Aspergillus flavus was the dominant species followed by Aspergillus parasiticus. Aflatoxin analyses of groundnut seed samples were performed using ultra performance liquid chromatography; 22.5% and 41.3% of samples were positive, with total aflatoxin concentrations of 786 and 3135 ng g-1 from 2013/2014 and 2014/2015 samples, respectively. The level of specific aflatoxin concentration varied between 0.1 and 2526 ng g-1 for B2 and B1, respectively. Among contaminated samples of groundnut cake, 68% exhibited aflatoxin concentration below 20 ng g-1, while as high as 158 ng g-1 aflatoxin B1 was recorded. The study confirms high contamination of groundnut products in East Ethiopia.

Orphan legume crops enter the genomics era!

Many of the world's most important food legumes are grown in arid and semi-arid regions of Africa and Asia, where crop productivity is hampered by biotic and abiotic stresses. Until recently, these crops have also suffered from a dearth of genomic and molecular-genetic resources and thus were 'orphans' of the genome revolution. However, the community of legume researchers has begun a concerted effort to change this situation. The driving force is a series of international collaborations that benefit from recent advances in genome sequencing and genotyping technologies. The focus of these activities is the development of genome-scale data sets that can be used in high-throughput approaches to facilitate genomics-assisted breeding in these legumes.

An integrated pipeline of open source software adapted for multi-CPU architectures: use in the large-scale identification of single nucleotide polymorphisms.

The large amounts of EST sequence data available from a single species of an organism as well as for several species within a genus provide an easy source of identification of intra- and interspecies single nucleotide polymorphisms (SNPs). In the case of model organisms, the data available are numerous, given the degree of redundancy in the deposited EST data. There are several available bioinformatics tools that can be used to mine this data; however, using them requires a certain level of expertise: the tools have to be used sequentially with accompanying format conversion and steps like clustering and assembly of sequences become time-intensive jobs even for moderately sized datasets. We report here a pipeline of open source software extended to run on multiple CPU architectures that can be used to mine large EST datasets for SNPs and identify restriction sites for assaying the SNPs so that cost-effective CAPS assays can be developed for SNP genotyping in genetics and breeding applications. At the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), the pipeline has been implemented to run on a Paracel high-performance system consisting of four dual AMD Opteron processors running Linux with MPICH. The pipeline can be accessed through user-friendly web interfaces at http://hpc.icrisat.cgiar.org/PBSWeb and is available on request for academic use. We have validated the developed pipeline by mining chickpea ESTs for interspecies SNPs, development of CAPS assays for SNP genotyping, and confirmation of restriction digestion pattern at the sequence level.

A database of annotated tentative orthologs from crop abiotic stress transcripts.

A minimal requirement to initiate a comparative genomics study on plant responses to abiotic stresses is a dataset of orthologous sequences. The availability of a large amount of sequence information, including those derived from stress cDNA libraries allow for the identification of stress related genes and orthologs associated with the stress response. Orthologous sequences serve as tools to explore genes and their relationships across species. For this purpose, ESTs from stress cDNA libraries across 16 crop species including 6 important cereal crops and 10 dicots were systematically collated and subjected to bioinformatics analysis such as clustering, grouping of tentative orthologous sets, identification of protein motifs/patterns in the predicted protein sequence, and annotation with stress conditions, tissue/library source and putative function. All data are available to the scientific community at http://intranet.icrisat.org/gt1/tog/homepage.htm. We believe that the availability of annotated plant abiotic stress ortholog sets will be a valuable resource for researchers studying the biology of environmental stresses in plant systems, molecular evolution and genomics.

A database of simple sequence repeats from cereal and legume expressed sequence tags mined in silico: survey and evaluation.

Simple sequence repeats (SSRs) or microsatellites are an important class of molecular markers for genome analysis and plant breeding applications. In this paper, the SSR distributions within ESTs from the legumes soybean (Glycine max, representing 135.86 Mb), medicago (Medicago truncatula, 121.1 Mb) and lotus (Lotus japonicus, 45.4 Mb) have been studied relative to the distributions in cereals such as sorghum (Sorghum bicolor, 98.9 Mb), rice (Oryza sativa, 143.9 Mb) and maize (Zea mays, 183.7 Mb). The relative abundance, density, composition and putative annotations of di-, tri-, tetra- and penta-nucleotide repeats have been compared and SSR containing ESTs (SSR-ESTs) have been clustered to give a non-redundant set of EST-SSRs, available in a database. Further, a subset of such candidate EST-SSRs from sorghum have been tested for their ability to detect polymorphism between Striga-susceptible, stay-green drought tolerant mapping population parent 'E 36-1' and its Striga-resistant, non-stay-green counterpart 'N13'. Primer sets for 64% of the EST-SSRs tested produced a clear and specific PCR product band and 34% of these detected scorable polymorphism between the N13 and E 36-1 parental lines. Over half of these markers have been genotyped on 94 RILs from the (N13 x E 36-1)-based mapping population, with 42 markers mapping onto the ten sorghum linkage groups. This establishes the value of this database as a resource of molecular markers for practical applications in cereal and legume genetics and breeding. The primer pairs for non-redundant EST-SSRs have been designed and are freely available through the database (http://intranet.icrisat.org/gt1/ssr/ssrdatabase.html).

A new intervarietal linkage map and its application for quantitative trait locus analysis of "gigas" features in bread wheat.

A doubled-haploid (DH) population from an intervarietal cross between the Japanese cultivar 'Fukuho-komugi' and the Israeli wheat line 'Oligoculm' was produced by means of wheat x maize crosses. One hundred seven DH lines were genotyped to construct a simple sequence repeat (SSR) based linkage map with RFLP, RAPD, and inter-simple sequence repeat markers. Out of 570 loci genotyped, 330 were chosen based on their positions on the linkage map to create a "framework" map for quantitative trait locus (QTL) analysis. Among the 28 linkage groups identified, 25 were assigned to the 21 chromosomes of wheat. The total map length was 3948 cM, including the three unassigned linkage groups (88 cM), and the mean interval between loci was 12.0 cM. Loci with segregation distortion were clustered on chromosomes 1A, 4B, 4D, 5A, 6A, 6B, and 6D. After vernalization, the DH lines were evaluated for spike number per plant (SN) and spike length (SL) in a greenhouse under 24-h daylength to assess the "gigas" features (extremely large spikes and leaves) of 'Oligoculm'. The DH lines were also autumn-sown in the field in two seasons (1990-1991 and 1997-1998) for SN and SL evaluation. QTL analysis was performed by composite interval mapping (CIM) with the framework map to detect QTLs for SN and SL. A major QTL on 1AS, which was stable in both greenhouse and field conditions, was found to control SN. This QTL was close to the glume pubescence locus (Hg) and explained up to 62.9% of the total phenotypic variation. The 'Oligoculm' allele restricted spike number. The SSR locus Xpsp2999 was the closest locus to this QTL and is considered to be a possible marker for restricted tillering derived from 'Oligoculm'. Eight QTLs were detected for SL. The largest QTL detected on 2DS was common to the greenhouse and field environments. It explained up to 33.3% of the total phenotypic variation. The second largest QTL on 1AS was common to the greenhouse and the 1997-1998 season. The position of this QTL was close to that for the SN detected on 1AS. The association between SN and SL is discussed.